Ssb method

Abstract

The invention relates to a method of characterising a target polynucleotide using a single-stranded binding protein (SSB). The SSB is either an SSB comprising a carboxy-terminal (C-terminal) region which does not have a net negative charge or a modified SSB comprising one or more modifications in its C-terminal region which decreases the net negative charge of the C-terminal region.

1 .- 38 . (canceled) 
     
     
         39 . A method of unblocking a transmembrane protein pore, the method comprising:
 (i) contacting the transmembrane protein pore with a polypeptide under an applied electrical potential;   (ii) measuring a current through the transmembrane protein pore indicative of blocking of the transmembrane protein pore by the polypeptide; and   (iii) reversing the applied electrical potential to clear the polypeptide from the transmembrane protein pore, thereby unblocking the transmembrane protein pore.   
     
     
         40 . The method of  claim 39 , wherein the polypeptide comprises a charged tail that contacts the transmembrane protein pore. 
     
     
         41 . The method of  claim 39 , wherein the polypeptide is a protein. 
     
     
         42 . The method of  claim 41 , wherein the protein is a single-stranded binding protein (SSB). 
     
     
         43 . The method of  claim 39 , wherein measuring a current through the transmembrane protein pore comprises measuring an ion current. 
     
     
         44 . The method of  claim 39 , wherein the applied electrical potential is a voltage potential. 
     
     
         45 . The method of  claim 44 , wherein the voltage potential is in the range of −400 mV to +400 mV. 
     
     
         46 . The method of  claim 39 , wherein reversing the applied electrical potential comprises applying a cycle of a period of a positive holding potential followed by a period of a negative holding potential. 
     
     
         47 . The method of  claim 46 , wherein the negative holding potential is followed by a rest period where no potential is applied. 
     
     
         48 . The method of  claim 39 , wherein the transmembrane protein pore is provided in an array of individually addressable transmembrane protein pores. 
     
     
         49 . The method of  claim 39 , wherein the transmembrane protein pore is selected from hemolysin, leukocidin,  Mycobacterium smegmatis  porin A (MspA), MspB, MspC, MspD, outer membrane phospholipase A,  Neisseria  autotransporter lipoprotein (NaIP) and WZA. 
     
     
         50 . The method of  claim 39 , wherein the barrel or channel of the transmembrane protein pore has a diameter of less than 7 nm at its narrowest point.